Summary of Work: The study of oxidative stress has attracted considerable interest and has been the focus of much research in recent years. Cumulative oxidative damage to tissues has been implicated in a number of disease states, e.g. the aging process, cancer, and ischemia reperfusion. The study of oxidative stress in the mitochondria has shown that hydrogen peroxide is produced via the incomplete reduction of oxygen during oxidative phosphorylation. Hydrogen peroxide levels are kept relatively low under normal physiological conditions. Under certain conditions, such as inflammation, excessive amounts of hydrogen peroxide are produced. The production of excess hydrogen peroxide is thought to precede several occurrences, such as lipid peroxidation, DNA and/or protein damage, and glutathione depletion, that are characteristic of oxidative stress. We are currently probing the role of cytochrome C free-radicals in oxidative stress by probing the anaerobic reaction between horse heart cytochrome C (HHCC) and hydrogen peroxide using the spin-trapping reagent 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The ESR spectrum of the reaction showed the presence of one major free-radical product with possibly a minor species also being formed, while the MS clearly showed the presence of at least four distinct DBNBS adducts. Enzymatic digest followed by LC/ESI/MS showed an ion due to the DBNBS adduct with Tyr74 of the HHCC which is on the surface of the protein. It is thought that the heme is initially oxidized and then the radical site is transferred intramolecularly to the tyrosine. We have also investigated the possibility that the oxidatively-induced free-radical on HHCC can initiate free-radical oxidation of other proteins that do not, by themselves, undergo free-radical oxidation under identical reaction conditions. We have found that proteins such as RNase A, oxidized insulin B and renin substrate tetradecapeptide, are readily oxidized by the HHCC free-radical. Proteolysis combined with MALDI/MS indicated that the DBNBS is located on a tyrosine residue of oxidized insulin B. Similarly, the radical site on RSTP has been identified as tyrosine 4 and the radical site on the nonapeptide RWIILGLNK has been unequivocally identified as the tryptophan. Addition of ascorbic acid has also been shown to prevent transfer of the radical from cytochrome c to other peptides.